Method of inhibiting thrombosis and complications after angioplasty using magnesium-based compound

ABSTRACT

Treatment with magnesium produces a inhibition of acute stent thrombosis under high-shear flow conditions without any hemostatic or significant hemodynamic complications.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to methods of treating thrombosis, andmore particularly to the use of magnesium to prevent conditions such asin-stent thrombosis.

[0003] 2. Discussion of the Related Art:

[0004] Coronary artery disease is one of the country's largest healthconcerns. According to the American Heart Association, this diseaseaffects 13.5 million Americans. Almost a million of these people haveexperienced heart attacks. Still others have experienced angina,undergone coronary artery bypass surgery and/or had heart transplants.Others in the later, or more severe, stages of coronary artery diseaseare in varying stages of congestive heart failure.

[0005] Coronary artery disease, which has been linked with the increasein cholesterol and saturated fat in our diets, is treatable. One mainsymptom of coronary artery disease is the deposit of these fattymaterials alongside a vessel wall such as an arterial wall. This resultsin the progressive narrowing of the lumen, and arteriosclerosis. Suchdeposits may be treated through a procedure called angioplasty. Duringangioplasty, the doctor inserts a catheter into an artery, typically agroin artery, and maneuvers the catheter up through the artery until thecatheter is positioned at the site of the narrowing or obstructioncaused by plaque. The plaque may then be flattened by inflating aballoon located around the tip of the catheter. As the balloon expands,it compresses the fatty deposits against the walls of the artery.

[0006] During this potentially lifesaving procedure, the doctor mayinsert a stent into the vessel at the site of the blockage. This small,typically metallic device helps to hold the vessel open and improvesblood flow. This serves to relieve the symptoms of Coronary ArteryDisease.

[0007] Unfortunately, stents do not provide an absolute solution to thisproblem. Restenosis, a narrowing of the passageway which may initiatedby platelet adhesion and aggregation at the site of arterial injury, andthrombosis frequently occur at the site of the stent. “Thrombosis”describes the formation of a thrombus, or blood clot, inside a bloodvessel. Thrombosis may be caused by the continuous stresses from bloodflow over the stent. The longitudinal lumen of a stent usually, if notalways, has an irregular surface or regions that protrude into the lumenthat can produce a turbulent fluid flow. Alternatively, the thrombosismay be the result of a foreign body reaction to the stent. The formationof a blood clot within a blood vessel may cause tissue damage. Such aclot may be life threatening, particularly when it partially orcompletely blocks the flow of blood through a blood vessel. Ifthrombosis occurs as a result of the stent placement, a secondaryprocedure or a surgical bypass operation is required.

[0008] Products such as aspirin, dipyridamole heparin and are known inthe art to dissolve such clots. While these products may eliminate theclot, they have the potential serious side effect of causing prolongedbleeding. Additionally, the effect of the administration of suchproducts may only be reversed by the formation or addition of newplatelets.

[0009] It is desired to be able to place a stent within a vessel throughpercutaneous transluminal delivery without the risk of in-stentthrombosis. Such stent placement without the risk of in-stent thrombosiswould greatly increase the quality of life for many patients.Accordingly, a method to prevent the formation of in-stent thrombosisthat is not detrimental to the patient is desired.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 illustrates a perfusion protocol that was run in an ex-vivoporcine shunt model according to an embodiment of the present;

[0011]FIG. 2(a) provides bar graphs showing the effect on thrombusweight in different treatment groups in an experiment according to oneembodiment of the present invention;

[0012]FIG. 2(b) provides views of nitinol stents (side-on view)depicting thrombus burden under the various treatment conditions;

[0013]FIG. 3 provides a graphical depiction of the effects of magnesiumon heart rate and mean arterial blood pressure as shown by an experimentthat was conducted according to one embodiment of the present invention;

[0014]FIG. 4 provides a chart detailing experimental data collected froman experiment run in accordance with one embodiment of the presentinvention; and

[0015]FIG. 5 provides a chart detailing the effects of the heparin andmagnesium as used in an experiment according to one embodiment of thepresent invention on 5 μg/mL collagen-induced platelet aggregation,bleeding time and ACT.

DETAILED DESCRIPTION

[0016] The present invention is the discovery that the administration ofmagnesium produces a significant time-dependent inhibition, andpotential prevention, of acute platelet-dependent stent thrombosis underhigh-shear flow conditions. This antithrombotic effect, which correlatesdirectly with serum magnesium ion levels, is primarily due to themagnesium's effect on platelets, magnesium's effect on coagulation andfibrinolysis.

[0017] An additional feature of the present invention is that theinhibition of acute platelet-dependent stent thrombosis may be achievedwithout any hemostatic or significant hemodynamic complications.Importantly, a significant inhibition of platelet-thrombus formation wasdemonstrated with magnesium treatment without any effect on plateletaggregation or bleeding time. The potent antithrombotic effects ofmagnesium together with its safety, ease of administration, and low costmake it a promising treatment during percutaneous coronary intervention(PCI).

[0018] The timing of the magnesium administration according to thepresent invention is important to the inhibition of stent thrombosis.The maximum antithrombotic effect of Mg is evident when platelets aretreated before exposure to thrombogenic stimuli. Such thrombogenicstimuli include, but are not limited to, catheters, endovasculardevices, stent-bearing catheters, angioplasty, stents, laser catheters,atherectomy, radiation, extraction devices (for example: transluminalextraction atherectomy), angiojets, and local drug-delivery catheters.

[0019] This time-dependent effect may be seen in FIG. 4, which providesdata from an experiment conducted in accordance with one embodiment ofthe present invention. As shown in FIG. 4, the antithrombotic effects ofmagnesium were significantly more pronounced when Mg was given 40minutes, as opposed to 20 minutes, before initiation of stent perfusion.Given sufficient exposure to magnesium with appropriate timing, theadministration of magnesium may be used to assist in the prevention ofstent thrombosis.

[0020] The effects of the present invention are best seen when themagnesium is administered between about 30 and 60 minutes prior tothrombotic stimuli. However, it should be understood that the magnesiummay be administered within a reasonable time outside of this range. Forexample, the magnesium may be administered between 15 and 90 minutesprior to thrombotic stimuli. The magnesium may be administered, at leastin part, by coating a stent or other endoprosthesis with amagnesium-containing compound prior to insertion into a body passageway.Administration of the magnesium should be continued for approximatelyeight to twelve hours after the procedure.

[0021] In one embodiment of the present invention, magnesium isadministered intravenously. Intravenous administration allows forgreater control of the level of magnesium in the blood. As should beunderstood by one skilled in the art, however, the administration ofmagnesium is not limited to intravenous administration. Other methods ofadministration include, but are not limited to, oral ingestion and theadministration of an injection. Oral administration of magnesium may beparticularly beneficial to treat (or prevent) stent thrombosis inpatients once they are discharged. The magnesium may alternatively besupplied to a patient through a catheter or other instrument which isinserted percutaneously, or through a tube attached to such instrument.Such an instrument may alternatively be coated with amagnesium-containing compound. In a further embodiment of the presentinvention, a stent may be coated with a magnesium-containing compound.

[0022] The use of magnesium disclosed herein has been thus far limitedto a discussion of the inhibition of stent thrombosis. However, thepresent invention is not so limited. Magnesium may be administered astaught by the present invention to prevent thrombosis during angioplastyand bypass and other surgeries.

[0023] The magnesium may be administered in the form of magnesiumsulfate (MgSO₄), or any other form of magnesium known by those skilledin the art to be non-toxic to the subject. Such non-toxic forms ofmagnesium include, but are not limited to, magnesium phosphate (MgPO₄),magnesium chloride (MgCl) and magnesium oxide (MgO).

[0024] Magnesium has an anti-adhesive effect on platelets that isachieved primarily by reducing calcium mobilization in platelets. It mayalso suppress fibrinogen interaction with platelets via competitiveinhibition of calcium at the calcium-binding sites of the glycoproteinIIb-IIIa complex. See Gawaz M, Ott I, Reininger A J, Neumann F J.Effects of magnesium on platelet aggregation and adhesion: magnesiummodulates surface expression of glycoproteins on platelets in vitro andex vivo. Thromb Haemost, 1994; 72:912-918. The serum Mg levels achievedin the example discussed herein (3.7±0.3 for heparin +Mg-early and3.8±0.4 for heparin+Mg-late) were in the range where the anti-adhesiveeffects would be more evident compared to the anti-aggregatory effect.

[0025] The discrepant effect on platelet adhesion/thrombus formation andplatelet aggregation may relate to the fact that the anti-adhesiveeffect of Mg may occur at a lower concentration (<4 mEq/L) compared tothe anti-aggregatory effect (>5 mEq/L). See Ravn H B, Kristensen S D,Hjortdal V E, Thygesen K, Husted S E, Early Administration ofIntravenous Magnesium Inhibits Arterial Thrombus Formation, ArteriosclerThromb Vasc Biol, 1997; 17:3620-3625; and Gawaz M, Ott I, Reininger A J,Neumann F J, Effects of magnesium on platelet aggregation and adhesion:magnesium modulates surface expression of glycoproteins on platelets invitro and ex vivo, Thromb Haemost, 1994; 72:912-918. Since the thrombusweights returned towards pre-treatment values during control perfusionruns post-treatment utilizing the same aortic strip, it is unlikely thatthe varying thrombogenicity of the porcine aortic strip may havecontributed to treatment effect.

[0026] The role that magnesium may take in the treatment ofcardiovascular diseases has not been determined. For example, recentclinical trials have presented conflicting evidence about the role ofmagnesium in acute myocardial infarction with the ISIS-4 trial(International Study of Infarct Survival) showing no benefit (ISIS-4: Arandomized factorial trial assessing early oral captopril, oralmononitrate, and intravenous magnesium sulfate in 58,050 patients withsuspected myocardial infarction. Lancet, 1995;345:669-685.) and theLimit-2 (Leicester Intravenous Magnesium Intervention Trial) studyproviding strong evidence for a survival advantage. See Woods K L,Fletcher S, Roffe C, Haider Y. Intravenous magnesium sulphate insuspected acute myocardial infarction: results of the second LeicesterIntravenous Magnesium Intervention Trial (Limit-2). Lancet,1992:339:1553-8.

[0027] Experimental Model

[0028] An experiment according to one embodiment of the presentinvention was performed using an ex-vivo model that primarily examinesshear-mediated, platelet-dependent thrombus formation. This experimentallowed for the evaluation of the effects of magnesium, and thetime-dependent nature thereof, on acute platelet-dependent stentthrombosis in an ex-vivo porcine arteriovenous shunt model of high-shearblood flow.

[0029] Such a model is useful to study interaction of blood elementswith stents and thrombogenic surfaces under controlled and well-definedconditions. This ex-vivo system was chosen for its reproducibility andsimplicity. It a sensitive tool to assess pre-clinical efficacy ofantithrombotic therapeutic interventions.

[0030] Animal Surgery

[0031] All procedures of the animal surgery conducted in conjunctionwith the experiments discussed herein were performed in accordance withone embodiment of the present invention were approved by theInstitutional Animal Care and Use Committee and conformed to theAmerican Heart Association guidelines for animal research. Experimentswere performed in 10 swine weighing 25 to 30 kg. After overnightfasting, swine were sedated with phenobarbital (5 mg/kg), and anesthesiawas maintained with 1% isoflurane after endotracheal intubation. Theright carotid artery and jugular vein were isolated and cannulated with8F sheaths to establish an extracorporeal circuit as describedpreviously. See Kaul S, Makkar R R, Nakamura M, Litvack F, Shah P K,Forrester J S, Hutsell T, Eigler N L, Inhibition of Acute StentThrombosis under High-Shear Flow Conditions by a Nitric Oxide Donor.DMHD/NO: An Ex-Vivo Porcine Arteriovenous Shunt Study, Circulation,1996, 94:2228-34. Arterial blood gases and pH were monitoredperiodically and maintained at normal levels by adjustment of theventilation rate and tidal volume. Invasive arterial pressuremeasurement, oxygen saturation, ECG, and rectal temperature weremonitored continuously. A thermostatically controlled blanket was usedto maintain temperature at 37° C. Venous blood was collected forbaseline platelet aggregation, complete blood cell count, and activatedclotting time (ACT) measurements.

[0032] All animals received heparin at a dose of 10 U/kg as a bolusbefore the study to prevent thrombotic occlusion of catheters andtubing. Each swine received an average of 200 U heparin, an amount thatproduces negligible effects on thrombus formation at high-shearconditions in this model. See Kaul S, Makkar R R, Nakamura M, Litvack F,Shah P K, Forrester J S, Hutsell T, Eigler N L, Inhibition of AcuteStent Thrombosis under High-Shear Flow Conditions by a Nitric OxideDonor. DMHD/NO: An Ex-Vivo Porcine Arteriovenous Shunt Study.Circulation, 1996, 94:2228-34. At the conclusion of the experiment,blood was collected for complete blood cell counts, the carotid arteryand jugular vein were ligated, and the animals were allowed to recoverfrom anesthesia before being returned to the vivarium. Each animal wasstudied twice with a minimum interval of 2 weeks between eachexperiment.

[0033] Coronary Stents

[0034] The stents tested were 7-mm-long slotted-tube-geometry devicesmade from the nickel-titanium alloy NITINOL (Advanced CoronaryTechnology, Menlo Park, Calif.) (n=156 stents in 10 swine). Each stentweighed 24±3 mg and had a strut thickness of 0.006 in. They had asilicon carbide grit-blasted surface finish, which creates a uniformroughened surface known to be highly thrombogenic in this model. SeeKaul S, Makkar R R, Nakamura M, Litvack F, Shah P K, Forrester J S,Hutsell T, Eigler N L, Inhibition of Acute Stent Thrombosis underHigh-Shear Flow Conditions by a Nitric Oxide Donor. DMHD/NO: An Ex-VivoPorcine Arteriovenous Shunt Study. Circulation, 1996, 94:2228-34; andMakkar R R, Eigler N L, Kaul S, Nakamura M, Forrester J S, Herbert J M,Litvack F I, Effects of clopidogrel, aspirin and combined therapy in aporcine ex vivo model of high-shear induced stent thrombosis, Eur HeartJ, 1998; 19:1538-1546. Stents were expanded on a tapered mandrel to anopen diameter of 2.0 mm before being mounted in the perfusion chamber.

[0035] Extracorporeal Shunt and Perfusion Protocol

[0036] The extracorporeal shunt system utilized in this study has beenextensively characterized and described previously. See Kaul S, Makkar RR, Nakamura M, Litvack F, Shah P K, Forrester J S, Hutsell T, Eigler NL, Inhibition of Acute Stent Thrombosis under High-Shear Flow Conditionsby a Nitric Oxide Donor. DMHD/NO: An Ex-Vivo Porcine Arteriovenous ShuntStudy, Circulation, 1996, 94:2228-34; Makkar R R, Litvack F, Eigler N L,Nakamura M, Ivey P A, Forrester J S, Shah P K, Jordan R E, Kaul S,Effects of GP IIb/IIIa Receptor Monoclonal Antibody (7E3), Heparin, andAspirin in an Ex Vivo Canine Arteriovenous Shunt Model of StentThrombosis, Circulation, 1997; 95(4): 1015-1021; and Makkar R R, EiglerN L, Kaul S, Nakamura M, Forrester J S, Herbert J-M, Litvack F I,Effects of clopidogrel, aspirin and combined therapy in a porcine exvivo model of high-shear induced stent thrombosis, Eur Heart J, 1998;19:1538-1546. After a 60-minute stabilization period, stents weremounted in the tubular chamber and perfused with normal saline for 60seconds at 37° C. With a switch valve used to prevent stasis, blood wascirculated through the system, and flow was regulated at 100 mL/min for20 minutes by using a peristaltic pump (Masterfiex, Cole-PalmerInstrument Co.) placed in the circuit distal to the perfusion chamber.

[0037] The selected flow rate generates a wall shear rate of 2100 s⁻¹ atthe stent surface. Shear rates were calculated according to the formulafor laminar flow of homogeneous Newtonian fluid in a cylindrical tube:shear rate=4.Q/π·R3, where Q is volume flow and R is radius. SeeGoldsmith H L, Turitto V T, Rheological aspects of thrombosis andhemostasis: basic principles and applications, Thromb Haemost,1986;55:415-435. At high shear rates, as used in this study, blood isconsidered to be essentially a Newtonian fluid.

[0038] At the end of the perfusion period, saline was circulated throughthe chamber and ex vivo system for several minutes at 40 mL/min to clearany visible blood before another stent was mounted. At the completion ofeach perfusion period, the stents (weighed prior to perfusion) wereremoved from the chamber, dried, and weighed immediately. Thrombusweight was calculated as a difference between pre- and post-perfusionstent weights.

[0039] The top of each stent was covered with a heterologous porcineaortic strip (Pel Freeze, Kans.) from which the intimal layer wasremoved to simulate the thrombogenic conditions induced by vascularinjury associated with stent implantation. The number of stent perfusionruns examined varied from 6 to 8 during each experiment. Digital imagesof stents were obtained with a Nikon 950 digital camera, downloaded intoa PC and processed with image analysis software (PhotoShop Adobe 5.0). Asample of such images may be seen in FIG. 2(b).

[0040] At the end of each treatment, control stents were perfused toensure return of stent thrombus weights towards baseline pre-treatmentvalues. Effects on thrombus weight (TW), whole-blood plateletaggregation (PA), bleeding time (BT), activated clotting time (ACT),serum magnesium level, and complete blood count (CBC) were quantified atvarious time points as shown in the protocol schematic. Mean arterialblood pressure (MABP) and heart rate (HR) were monitored and recordedthroughout the protocol.

[0041] Thirty minutes after administration of the heparin (in theheparin alone) or magnesium, 3 mL venous blood was collected in asiliconized test tube containing 0.3 mL of 0.129 molar sodium citrate orsodium heparin (Becton Dickinson Vacutainer System). Whole bloodaggregometry (Chronolog Corp.) was used to measure collagen (2 and 5μg/mL)- and Adenosine diphosphate (2.5 μM)-induced platelet aggregation.Aggregation was expressed as maximal increase in electrical impedancemeasured in ohms at 6 minutes after the addition of agonist. Thebaseline platelet aggregation was 25±3 ohms. The platelet aggregationafter the addition of Mg-late alone and heparin alone was 22±3 and 24±6ohms, respectively. As noted above, data points in the Mg-treatedanimals were examined within 20 minutes post-bolus (Mg-early) and >40minutes post-bolus (Mg-late). The platelet aggregation obtained within20 minutes post-bolus was 23±4 ohms. The platelet aggregationobtained >40 minutes post-bolus was 21.0±5 ohms.

[0042] Bleeding time is defined as the time from the creation of anincision to the point where bleeding from the incision ceases. In theexperiment according to one embodiment of the present inventiondiscussed herein, bleeding time was measured from an incision on theventral surface of the thigh with a No. 11 surgical knife.

[0043] In the experiment discussed herein that was conducted accordingto one embodiment of the present invention, ACT was determined using aHemochron 400 (International Technidyne Corp.) machine in standardfashion. See Makkar R R, Litvack F, Eigler N L, Nakamura M, Ivey P A,Forrester J S, Shah P K, Jordan R E, Kaul S, Effects of GP IIb/IIIaReceptor Monoclonal Antibody (7E3), Heparin, and Aspirin in an Ex VivoCanine Arteriovenous Shunt Model of Stent Thrombosis, Circulation, 1997;95(4): 1015-1021; and Makkar R R, Eigler N L, Kaul S, Nakamura M,Forrester J S, Herbert J-M, Litvack F I, Effects of clopidogrel, aspirinand combined therapy in a porcine ex vivo model of high-shear inducedstent thrombosis, Eur Heart J, 1998; 19:1538-1546.

[0044] Serum magnesium levels for the test subjects were measuredspectrophotometrically using the magon dye method. See Elm R J,Determination of serum magnesium concentration by clinical laboratories,Magnes Trace Elem, 1991-92:60-6.

[0045]FIG. 1 illustrates a perfusion protocol that was run in an ex-vivoporcine shunt model according to an embodiment of the present invention.As used in FIG. 1, PA means platelet aggregation, ACT means activatedclotting time, BP means arterial blood pressure, and CBC means completeblood count. In order to obtain control thrombus weight, two to threestents were perfused in each animal prior to the administration of anydrug.

[0046] The stents were perfused pre- and post-treatment with heparin ormagnesium in a random fashion. Heparin was administered as 50 U/kg IVbolus followed by 25-50 U/kg/hr IV to keep ACT >150 seconds. Magnesiumsulfate (MgS04) was administered as a 2 gm bolus IV over 20 minutesfollowed by 2 gm/hour maintenance infusion. To assess a potentialtime-dependent antithrombotic effect of magnesium, data points wereexamined within 20 minutes post-bolus (Mg-early) and >40 minutespost-bolus (Mg-late). The time-dependent effects of magnesium were alsoexamined in a random fashion.

[0047] As shown in FIG. 1, the “pre-treatment” readings were taken froma stent that was expanded to 2 mm in diameter in a tubular perfusionchamber interposed in the shunt and perfused with blood at a shear rateof 2100 s⁻¹ for 20 minutes, without any magnesium or heparin in thesystem. The “heparin alone” readings were taken from stents that wereperfused with blood for 20 minutes when the animals had been treatedwith intravenous heparin as described above. The perfusion study for the“heparin alone” reading was performed between twenty and thirty minutesafter the administration of heparin. Both the “heparin+Mg-Early” and“heparin+Mg-Late” readings were taken from stents that were perfusedwith blood for 20 minutes after the animals had been treated withintravenous heparin and magnesium as noted above. The perfusion periodfor the heparin+Mg-early readings started within 20 minutes afteradministration of the MgSO₄ bolus. The perfusion period for the heparin+Mg-late readings started more than 40 minutes after the administrationof the MgSO₄ bolus.

[0048] In one embodiment of the present invention, the magnesium bolusis followed by a 1 to 2 gm/hour maintenance infusion. For example, inthe experiment discussed in FIG. 1 herein, a 2 gm bolus IV wasadministered to the test subject over 20 minutes followed by a 2 gm/hourmaintenance infusion for an average magnesium dosage of 9 gm in a 30 kgpig (the dosages ranged from 5-13 gm).

[0049] The magnesium dosage required to achieve the benefits of oneembodiment of the present invention is generally between 0.16 gm and 0.4gm per kilogram body weight. A human weighing 100-kg would preferablyintravenously receive an approximately 2 gm bolus IV for roughly 10-20minutes. This would preferably be followed by an approximately 2 gm/hourmaintenance infusion for roughly 8-12 hours. It should be understood byone skilled in the art, however, that these dosages and times ofadministration may be varied depending on a number of factors including,but not limited to, the size of the subject. That is, a bolus dose of1-4 gm over 10-20 minutes may be administered, followed by a maintenancedose of 1-4 gm/hr. It should be noted that at higher bolus andmaintenance doses greater anti-thrombotic effects are observed. However,higher dosages also tend to affect heart rate and blood pressure.

[0050]FIG. 2(a) provides a bar graph showing the effect on thrombusweight in different treatment groups in an experiment according to oneembodiment of the present invention. FIG. 2(b) provides side-on views ofnitinol stents depicting thrombus burden under the various treatmentconditions. Values are given as mean±SD. Eight to 15 stents were testedin the Magnesium alone groups. The number of stents varied between 20and 35.

[0051] As may be seen from FIG. 2(a), the category of stents labeled“pre-treatment” has the greatest amount of stent thrombosis. Magnesiumalone (both early and late groups) as well as heparin reduce thrombusformation slightly. Heparin with magnesium-early reduces the thrombosismore significantly, and heparin with magnesium-late virtually preventedthrombus formation.

[0052]FIG. 3 provides a graphical depiction of the effects according toone embodiment of the present invention of magnesium on heart rate andmean arterial blood pressure (MABP). In this experiment, magnesium inthe form of magnesium sulfate was administered intravenously to thesubject animals. Values are again provided as mean+SD. As may be seenfrom FIG. 3, magnesium had no statistically significant effects oneither heart rate or MABP.

[0053] The graph in FIG. 3 was generated by taking between 10 and 14observations at each time point. The MABP was measured in millimeters ofmercury (mmHg). The heart rate was measured in beats per minute (BPM).As may be seen from the graph, no statistically significant changes inheart rate and blood pressure were observed using ANOVA.

[0054]FIG. 4 provides a chart detailing the reduction in stentthrombosis based on five differing stimuli according to one embodimentof the present invention. Particularly, FIG. 4 shows the effects oftreatment with magnesium alone, heparin alone as well as combinedtreatment with magnesium and heparin on acute stent thrombosis. The datashown in FIG. 4 was obtained from an experiment similar to that whichwas discussed in FIGS. 2(a) and (b).

[0055] The total weight of a stent that was perfused with blood withoutthe addition of heparin or magnesium was 20±4 mg is referred to as stentTW. As shown in FIG. 1, the “pre-treatment,” or baseline stent TW,readings were taken from a stent that was perfused with blood for 20minutes, without any magnesium or heparin in the system. This number wasused to calculate the reduction in thrombus formation. The reduction inthrombus formation was calculated by subtracting the weight of apost-treatment stent (post-heparin or magnesium treatment) from theaverage weight of a stent that had been perfused with blood without theaddition of heparin or magnesium.

[0056] As shown in FIG. 4, stent TW was reduced by 42+21%, 47+19%,48±16% in the Mg-early, Mg-late and heparin-treated group, respectively.The weight was reduced by 67±12% in the heparin+Mg-early-treated group.The thrombus weight was further reduced by 86±8% in theheparin+Mg-late-treated group. All five of these calculations had aP<0.001 versus pretreatment. (P<0.05 heparin+Mg-late versus heparin andMg-early; P<.01 heparin+Mg-early and Mg-late versus heparin alone,Mg-early and late alone, ANOVA). Magnesium had no significant effects onplatelet aggregation, activated clotting time and bleeding time. Theserum Mg level was the only variable that correlated with TW (r=−0.70.P.0.002). There were no significant effects on heart rate or meanarterial blood pressure.

[0057] As may be seen in FIG. 4, the antithrombotic effects of combinedtreatment with heparin and Mg were significantly more pronouncedcompared with magnesium or heparin alone (P<0.001, ANOVA).Heparin+Mg-late produced a slightly greater, and statisticallysignificant, reduction in TW compared with heparin+Mg-early group(P<0.05, ANOVA).

[0058]FIG. 5 provides a chart detailing the effects of the heparin andmagnesium as used in an experiment according to one embodiment of thepresent invention on 5 μg/mL collagen-induced platelet aggregation,bleeding time and ACTs.

[0059] As shown in FIG. 5, the reduction in whole-blood plateletaggregation with the addition of magnesium, from 25±3 to 21±5 ohms, wasnot significant. To exclude the possibility that minimal magnesiumeffect on platelet aggregation may be related to the citrateanticoagulant used which may influence ionic concentrations because ofcalcium chelation in the samples, aggregation was tested in heparinizedas well as citrated blood in 3 pigs. Magnesium produced no significantinhibitory effect on platelet aggregation in heparin-stabilized samples(26±2 pre- and 27±2 post-Mg) as compared to citrate-stabilized blood(25±3 pre- and 22±3 post-Mg). Platelet aggregation was, however,slightly enhanced in heparinized samples. There was no effect ofmagnesium on platelet aggregation in response to either a lowerconcentration of collagen—2 μg/ml or to another platelet agonist—ADP(2.5 μM) (data not shown). Therefore, magnesium had no significanteffect on platelet aggregation, regardless of the anticoagulant used tostabilize blood.

[0060] As shown in FIG. 5, heparin increased the bleeding time from4.0±0.5 to 5.3±0.6. Heparin also prolonged ACT from 109±8 seconds to185±36 seconds (P<0.01. ANOVA). Magnesium had no significant effects oneither bleeding time or ACT beyond the heparin effect. There were noepisodes of significant bleeding in any of the animals studied.Treatment with heparin or magnesium had no significant effects on eitherplatelet or white blood cell counts or hematocrit (data not shown).

[0061] As further shown in FIG. 5, serum magnesium levels were higher inthe magnesium-treated animals compared to control. However, the levelswere virtually similar in the Mg-early and Mg-late group. Serummagnesium correlates significantly with thrombus weight (r=−0.70.P=0.002), bleeding time (r=0.54, P=0.05) and heart rate (r=−0.55,P=0.002). Serum magnesium was the only variable that correlatedsignificantly with thrombus weigh. No significant correlation ofthrombus weight was observed with platelet aggregation (r=0.4, P=0.07).

[0062] As previously noted, the timing of the administration ofmagnesium weighs significantly on the benefit seen in reduced thrombosisweight. Heparin+Mg-early produced a significant reduction in thrombosisweight when compared to the thrombus weight for heparin alone or thepre-treatment group. However, heparin+Mg-late produced an even greaterreduction in thrombosis weight. The difference in antithrombotic effectin Mg-early and Mg-late group cannot be solely explained by adose-dependent effect since serum magnesium levels were notsignificantly different. However, serum magnesium does not accuratelyreflect intracellular levels of magnesium. Given the increased exposureto magnesium, it is likely that the intracellular levels of Mg may behigher in the Mg-late as compared with Mg-early tests despite similarserum Mg levels.

[0063] Human Testing

[0064] One embodiment of the present invention was tested in humansubjects, with very positive results. Based on these tests, the use ofmagnesium was determined to be beneficial to prevent in-stentthrombosis. This effect was evident without any hemostatic orhemodynamic complications.

[0065] Twenty one low-risk patients undergoing non-acute percutaneouscoronary intervention with stent implantation received a two gm bolus ofintravenous MgSO₄ followed by a 1.5 gm/hour infusion for four hours anda 1 gm/hour infusion for the next eight hours. This test resulted in atotal administration of 16 gm of magnesium. The preset primary endpointsfor this experiment were: acute thrombotic occlusion and need forplatelet IIb/IIIa inhibitors bailout, death, myocardial infarction,recurrent ischemia and need for urgent revascularization at 48 hours and30 days. The secondary safety endpoints included hypotension,bradycardia, bleeding complications and heart block within the first 24hours.

[0066] In all cases, the interventions were finished with normalcoronary artery blood flow (TIMI 3 flow). There was no case of GPIIb/IIIa inhibitors bailout. Death, myocardial infarction, and urgentrevascularization were also not seen. Serum magnesium levels increasedsignificantly from a 2.1±0.3 baseline to 3.2±0.3 mg/dL post-bolus(P<0.0001). Further, magnesium did not have a significant effect oneither heart rate or mean arterial blood pressure. There was no furthersignificant increase in magnesium concentration after completion of themagnesium sulfate infusion (the magnesium level post-infusion was3.5±0.8 mg/dL).

[0067] All of the data presented herein is presented as mean±SD. Thestatistical difference between means was determined by one-way ANOVA. Ifmeans were shown to be significantly different, multiple comparisons bypairs were performed by the Bonferroni test (Graphpad Prism version3.0). Spearman's correlation analysis was performed to explore therelationship between serum magnesium and other variables and betweenthrombus size and other variables. A value of P<0.05 was consideredsignificant.

[0068] While the description above refers to particular embodiments ofthe present invention, it will be understood that many modifications maybe made without departing from the spirit thereof. The accompanyingclaims are intended to cover such modifications as would fall within thetrue scope and spirit of the present invention. The presently disclosedembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims, rather than the foregoing description,and all changes that come within the meaning and range of equivalency ofthe claims are therefore intended to be embraced therein.

What is claimed is:
 1. A method of inhibiting stent thrombosis underhigh shear blood flow conditions, the method comprising administering aneffective amount of a magnesium-based compound to a subject betweenapproximately 15 and 90 minutes prior to exposure of the subject tothrombogenic stimuli.
 2. The method of claim 1, wherein themagnesium-based compound is selected from the group consisting ofmagnesium sulfate, magnesium oxide, and magnesium chloride.
 3. Themethod of claim 1, whereby the magnesium-based compound is administeredintravenously.
 4. The method of claim 1, whereby the magnesium-basedcompound is administered orally.
 5. The method of claim 1, whereby themagnesium-based compound is administered as an injection.
 6. The methodof claim 1, whereby the magnesium-based compound is administered to thesubject between 30 and 60 minutes prior to exposure of the subject tothrombogenic stimuli.
 7. The method of claim 1, further including thestep of inserting a stent into the cardiovascular region of the subject,thereby exposing the subject to thrombogenic stimuli.
 8. The method ofclaim 1, further including the step of administering an effective amountof heparin to the subject.
 9. A method of reducing stent thrombosisunder high shear blood flow conditions, the method comprising: coating astent with an effective amount of a magnesium-based compound; andinserting the stent within a body passageway.
 10. The method of claim 9,wherein the magnesium-based compound is selected from the groupconsisting of magnesium sulfate, magnesium oxide, and magnesiumchloride.
 11. The method of reducing stent thrombosis according to claim9, wherein the magnesium-based compound is administered intravenously.12. The method of claim 9, further including the step of associating thestent with an instrument for inserting the stent within a bodypassageway.
 13. The method of claim 12, wherein the instrument is coatedwith a magnesium-containing compound.
 14. The method of claim 9, furtherincluding the step of administering a magnesium-based compound to asubject so that a serum magnesium level between 3 and 5 mEq/L isattained prior to exposure of the subject to a stent-inducedthrombogenic stimuli, wherein the administering step is carried outprior to inserting the stent within the body passageway.
 15. The methodof reducing stent thrombosis according to claim 14, wherein the serummagnesium level is attained at least 15-20 minutes before the subject isexposed to the stent-induced thrombogenic stimuli.
 16. A method ofinhibiting thrombosis under high shear blood flow conditions, the methodcomprising administering a magnesium-based compound as a 2 gm bolusprior to exposure to thrombogenic stimuli, followed by administering a 1to 1.5 gm/hour maintenance infusion of a magnesium-based compound. 17.The method of claim 16, wherein the bolus is administered over a 10 to20 minute time period.
 18. The method of claim 16, wherein themaintenance infusion is administered after exposure to thrombogenicstimuli.
 19. The method of claim 16, wherein the maintenance infusion isadministered over an 8 to 12 hour time period.
 20. The method of claim16, wherein the magnesium-based compound is selected from the groupconsisting of magnesium sulfate, magnesium oxide, and magnesiumchloride.
 21. A medical instrument configured for insertion into a bodyvessel coated with a magnesium-containing compound in an amounteffective to reduce the incidence of thrombosis in a cardiac region. 22.The medical instrument as in claim 21, wherein the medical instrument isa stent configured for insertion into the cardiac region.
 23. Themedical instrument as in claim 21, wherein the medical instrument is acatheter configured for insertion into the cardiac region.
 24. Themedical instrument as in claim 21, wherein the magnesium-containingcompound is selected from the group consisting of magnesium sulfate,magnesium oxide, and magnesium chloride.